ABSTRACT
The history of the topic of proteins at soft interfaces dates back to the 19th century, and until the present day, it has continuously attracted great scientific interest. A multitude of experimental methods and theoretical approaches have been developed to serve the research progress in this large domain of colloid and interface science, including the area of soft colloids such as foams and emulsions. From classical methods like surface tension adsorption isotherms, surface pressure-area measurements for spread layers, and surface rheology probing the dynamics of adsorption, nowadays, advanced surface-sensitive techniques based on spectroscopy, microscopy, and the reflection of light, X-rays and neutrons at liquid/fluid interfaces offers important complementary sources of information. Apart from the fundamental characteristics of protein adsorption layers, i.e., surface tension and surface excess, the nanoscale structure of such layers and the interfacial protein conformations and morphologies are of pivotal importance for extending the depth of understanding on the topic. In this review article, we provide an extensive overview of the application of three methods, namely, ellipsometry, X-ray reflectometry and neutron reflectometry, for adsorption and structural studies on proteins at water/air and water/oil interfaces. The main attention is placed on the development of experimental approaches and on a discussion of the relevant achievements in terms of notable experimental results. We have attempted to cover the whole history of protein studies with these techniques, and thus, we believe the review should serve as a valuable reference to fuel ideas for a wide spectrum of researchers in different scientific fields where proteins at soft interface may be of relevance.
Subject(s)
Proteins , Proteins/chemistry , Adsorption , Surface Properties , Water/chemistry , Colloids/chemistryABSTRACT
The effect of the degree of isotopic substitution of the aqueous medium on the adsorption kinetics and the surface dilational rheological behavior at the water/air interface of the globular protein ß-lactoglobulin was investigated. Aqueous solutions with fixed concentrations of 1 µM protein and 10 mM hydrogenous buffer with controlled pH 7 were prepared in H2O, D2O, and an isotopic mixture of 8.1% v/v D2O in H2O (called air contrast matched water, ACMW). Using a bubble shape analysis tensiometer, we obtained various experimental dependencies of the dilational viscoelasticity modulus E as a function of the dynamic surface pressure and of the frequency and amplitude of bubble surface area oscillations, either in the course of adsorption or after having reached a steady state. In general, the results revealed virtually no effect from substituting H2O by ACMW but distinct albeit relatively weak effects for intermediate adsorption times for D2O as the aqueous phase. In the final stage of adsorption, established after around 10 h, the equilibrium adsorption and the dilational rheological behavior of all protein layers under investigation are only very weakly affected by the presence of D2O. The obtained results help to design experimental protocols for protein adsorption studies, for example, by neutron reflectivity.
ABSTRACT
Protein adsorption to surfaces is at the heart of numerous technological and bioanalytical applications, but sometimes, it is also associated with medical risks. To deepen our insights into processes involving layers of surface-adsorbed proteins, high-resolution structural information is essential. Here, we use standing-wave X-ray fluorescence (SWXF) in combination with an optimized liquid-cell setup to investigate the underwater conformation of the random-coiled phosphoprotein ß-casein adsorbed to hydrophilic and hydrophobized solid surfaces. The orientation of the protein, as determined through the distributions of sulfur and phosphorus, is found to be sensitive to the chemical nature of the substrate. While no preferred orientations are observed on hydrophobized surfaces, on hydrophilic Al oxide, ß-casein is adsorbed as a diblock copolymer with the phosphorylated domain I attached to the surface. Our results demonstrate that targeting biologically relevant chemical elements with SWXF enables a detailed investigation of biomolecular layers under near-physiological conditions.
Subject(s)
Membrane Proteins , Adsorption , Fluorescence , Protein Conformation , Surface Properties , X-RaysABSTRACT
Several characteristics of ß-lactoglobulin (BLG) layers adsorbed at the air/water interface exhibit a strong pH dependence, but our knowledge on the underlying structure-property relations is still fragmental. Here, we therefore extend our recent studies by neutron reflectometry (NR) and provide a comprehensive overview through direct measurements of the surface excess Γ and the layers' molecular structure. This enables comparison with available literature data to draw general conclusions. The NR experiments were performed at various pH values and within a wide range of protein concentrations, CBLG. Adsorption kinetics measurements in air-contrast-matched-water and over a narrow Qz range enabled direct quantification of the dynamic surface excess Γ(t) and are found to be consistent with ellipsometry data. Near the isoelectric point, pI, the rates of adsorption and Γ are maximal but only at sufficiently high CBLG. NR data collected over a wider Qz range and in two aqueous isotopic contrasts revealed the structure of adsorbed BLG layers at a steady state close to equilibrium. Independent of the pH, BLG was found to form dense monolayers with average thicknesses of 1.1 nm, suggesting flattening of the BLG globules upon adsorption as compared with their bulk dimensions (≈3.5 nm). Near pI and at sufficiently high CBLG, a thick (≈5.5 nm) but looser secondary sublayer is additionally formed adjacent to the dense primary monolayer. The thickness of this sublayer can be interpreted in terms of disordered BLG dimers. The results obtained and notably the specific interfacial structuring of BLG near pI complement previous observations relating the impact of solution pH and CBLG on other interfacial characteristics such as surface pressure and surface dilational viscoelasticity modulus.
Subject(s)
Air , Lactoglobulins/chemistry , Water/chemistry , Adsorption , Hydrogen-Ion Concentration , Neutron Diffraction , Surface PropertiesABSTRACT
The properties of proteins at interfaces are important to many processes as well as in soft matter materials such as aqueous foam. Particularly, the protein interfacial behavior is strongly linked to different factors like the solution pH or the presence of electrolytes. Here, the nature of the electrolyte ions can significantly modify the interfacial properties of proteins. Therefore, molecular level studies on interfacial structures and charging states are needed. In this work, we addressed the effects of Y3+ and Nd3+ cations on the adsorption of the whey protein ß-lactoglobulin (BLG) at air-water interfaces as the function of electrolyte concentration. Both cations caused very similar but dramatic changes at the interface and in the bulk solution. Here, measurements of the electrophoretic mobility and with vibrational sum-frequency generation (SFG) spectroscopy were applied and consistently showed a reversal of the BLG net charge at remarkably low ion concentrations of 30 (bulk) and 40 (interface) µM of Y3+ or Nd3+ for a BLG concentration of 15 µM. SFG spectra of carboxylate stretching vibrations from Asp or Glu residues of interfacial BLG showed significant changes in the resonance frequency, which we associate to specific and efficient binding of Y3+ or Nd3+ ions to the proteins carboxylate groups. Characteristic reentrant condensation for BLG moieties with bound trivalent ions was found in a broad concentration range around the point of zero net charge. The highest colloidal stability of BLG was found for ion concentrations <20 µM and >50 µM. Investigations on macroscopic foams from BLG solutions revealed the existence of structure-property relations between the interfacial charging state and the foam stability. In fact, a minimum in foam stability at 20 µM ion concentration was found when the interfacial net charge was negligible. At this concentration, we propose that the persistent BLG molecules and weakly charged BLG aggregates drive foam stability, while outside the bulk reentrant zone the electrostatic disjoining pressure inside foam lamellae dominates foam stability. Our results provide new information on the charge reversal at the liquid-gas interface of protein/ion dispersions. Therefore, we see our findings as an important step in the clarification of reentrant condensation effects at interfaces and their relevance to foam stability.